Abstract: Lipids play a central role in cell growth and homeostasis as they compartmentalize the cell and provide biochemical cues to signaling proteins that are both transmembrane and peripheral proteins. These lipid signals include glycerophospholipids such as the phosphoinositides and phosphatidylserine, sphingolipids such as sphingosine-1-phosphate (S1P), ceramide, and ceramide-1-phosphate (C1P), and sterols such as cholesterol. Aberrant signaling by these lipids can result in a multitude of diseases such as cancers and neurodegenerative diseases. Quantifying and tracking these lipid signals in real-time in live cell model systems has proved difficult but generation of fluorescently tagged lipid binding domains has proven useful in monitoring the localization of phosphatidylserine, phosphoinositides (including PI(3)P, PI(4)P, PI(4,5)P2, PI(3,4)P2, and PIP3), and cholesterol. Antibodies have been generated to allow fixed cell localization of phosphatidylserine, PI(4)P, PI(3)P, and phosphatidycholine among others. More recently, chemical modification of lipid-binding sensors has allowed quantification of phosphoinositides including PI(4,5)P2 and PIP3. Despite these advances, there is dearth of tools to track and quantify sphingolipids. Sphingolipids have key roles in cellular signaling and membrane trafficking with main players including sphingosine, S1P, ceramide, and C1P. Ceramide is at the center of sphingolipid metabolism and is produced via de novo synthesis by a catabolic pathway from sphingomyelin or by a salvage/sphingosine feedback pathway utilizing ceramide synthases. C1P a major metabolite of ceramide contains a phosphomonoester headgroup and in contrast to ceramide has been shown to regulate cell proliferation, serve as a pro-inflammatory signal, regulate apoptosis, and macrophage chemotaxis. Recently, antibodies against ceramide and S1P have been generated but no chemical or biological sensors of C1P have been implemented, despite a critical role of C1P in many diseases that effect humans. In response to this NIGMS R21 FOA, we hypothesize new technology can be explored for monitoring and mapping C1P in live human cell culture. We hypothesize a robust and unbiased fluorescent biosensor of C1P can be generated using rationale of known C1P binding proteins and efficiency of other well established biosensors of glycerophospholipids. This proposal will explore a new strategy for development of a viable in vitro and cellular biosensor of C1P. Taken together, this R21 will use strong rationale of known C1P biology and cellular localization along with knowledge of C1P-protein interactions to explore the most favorable strategies for detection and quantification of cellular fluctuations of C1P. !